Coryneform bacteria have been widely used to produce chemical substances which have various applications in industries of animal feed, pharmaceuticals, food, and the like including L-lysine, L-threonine and various nucleic acids. In order to develop high yield strains from such coryneform bacteria using genetic engineering and metabolic engineering techniques, expression of genes involved in various metabolic pathways in coryneform bacteria need to be selectively regulated, and thus promoters useful for these gene regulations are required.
Conventional methods of isolating promoters include: (1) a method of using promoter probe vector randomly cloning genomic DNA fragments upstream of a reporter gene expressed only when a cloned fragment contains promoter activity; (2) a method of isolating genes and their promoters from a gene library using gene-specific probe-based hybridization; and (3) a differential hybridization of a gene bank using an inducible cDNA probe and a non-inducible cDNA probe.
In the expression of genes in coryneform bacteria, genes are generally expressed under their control of original promoters (Vasicova, P., et al., J. Bacteriol., 181, 6188-6191, (1999), etc.). However, typical structures of promoter sequences for gene expressions in coryneform bacteria have been unknown unlike other industrial microorganisms such as Escherichia coli, Bacillus subtilis, and the like. Thus, promoters for use in coryneform bacteria have been developed by eliminating a promoter region from a gene associated with resistance to antibiotics such as chloramphenicol, introducing into the promoter site a chromosomal DNA fragment isolated from coryneform bacteria with suitable restriction digestion, transforming coryneform bacteria with the resulting DNA molecules, and assessing antibiotic resistance of obtained strains (Eikmanns, B. J., et al., Gene, 102, 93-98, (1991); Patek, M., et al., Microbiology, 142, 1297-1309, (1996)). However, conventionally developed promoter sequences still need to be improved with respect to selectivity of gene expression, expression efficiency of genes, etc.
We developed a novel promoter nucleic acid molecule derived from Corynebacterium glutamicum by searching and amplifying putative promoter regions by polymerase chain reaction (PCR), introducing the putative promoter into the initiation site of lysC gene lacking a promoter, and identifying variations in lysC activity via lysine production to select efficient promoters.